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Results: Nonrandom and overlapping distributions of the relevant markers were found in biopsy/cellblock and surgical specimens, which closely correlated with each other and the diverse tumor categories, with no differences in area under curve-receiver-operating-characteristic curves for each marker between any two samples, including p40 and p63. Diagnostic combinations were p40−/TTF1+ or TTF1− for AD (where p40 was negative, apart from 5/30 AD showing at the best 1–2% tumor cells with low intensity); p40+/TTF1− (p40 strong and by far higher than 50%) for SQC; and p40+/TTF1+ or p40+/TTF1− (p40 strong and less than 50%) for ADSQC. The single SC case was p40−/TTF1−, suggesting glandular lineage. Practically, 41/46 (89%) tumors were correctly classified by IHC on small samples, including 30 AD, 10 SQC, 1/5 ADSQC, and no SC. Underdiagnosis of ADSQC was actually because of sampling error of biopsies/cellblocks rather than insufficient biomarker robustness, whereas underdiagnosis of SC was really because of the failure of either marker to highlight epithelial-mesenchymal transition.

Conclusions: This minimalist IHC-based model of p40 and TTF1 on biopsy/cellblock samples was effective to correctly subtype most cases of lung cancer.

An emerging issue of the so-called therapeutic pathology1 in lung cancer regards the precise subtyping of non-small cell lung carcinomas (NSCLC),2 which may yet present a difficult challenge by morphology alone when dealing with limited material and/or poorly differentiated tumors.2–6 This issue is relevant to clinics, when considering that more than two-thirds of patients are still diagnosed late with locally advanced or metastatic diseases, for whom tailored therapy may be required.7 We recently have indicated that immunohistochemistry (IHC)-driven small biopsies paralleled ultimate profiling and diagnoses on surgical specimens, paving the way to its potential application to perspective clinical trials too on lung cancer.8 A paradigm shift of the traditional morphology-related approach on small cytology and/or biopsy specimens5,9 has recently been incorporated into the new IASLC/ATS/ERS classification on lung adenocarcinoma (AD), which recommends using IHC and/or multidisciplinary setting to minimize the category NSCLC-not otherwise specified (NOS) and sparing diagnostic material for predictive molecular testing.9–11 Hence, a minimalist IHC algorithm approach based on informative, reliable, reproducible, and easy-to-assess biomarkers is warmly advisable for NSCLC to be typed for the sake of clinical usefulness and tenability of costs.

We have hypothesized that, in small biopsy or cellblock samples, a two-hit diagnostic model based on thyroid transcription factor-1 (TTF1), a very popular and sensitive detector of pulmonary AD12–16 and p40, a relatively poorly known marker of squamous cell differentiation, either nonneoplastic or tumoral, related to nontransactivating (non-TA), truncated, or ΔN isoforms of the p63 gene family of nuclear transcription factors,17–19 could be helpful to better refine NSCLC diagnoses. The p63 gene encodes diverse mRNA isoforms, which are generated by the activity of two different promoters, one of which internal to exon 3, which lead to the accumulation of TAp63 isoforms acting as TA agents favoring cell cycle arrest with apoptosis and cell differentiation induction and/or ΔNp63-p40 isoforms acting as negative dominant agents, which stimulate cell proliferation and block apoptosis with unrestrained tumor cell growth.20 In the normal lung tissue, by IHC, ΔNp63-p40 is confined to the nonterminal respiratory branch21 where it consistently decorates the basal layer of the bronchial/bronchiolar epithelium and the myoepithelial cells of the bronchial glands, whereas TAp63 may be inconsistently expressed by type II pneumocytes especially in areas of fibrosis and inflammation.22

Although TTF1 is also shared by other pulmonary tumors, including neuroendocrine tumors,23–26 and, quite rarely, nonpulmonary and nonthyroidal tumors,27 p40 expression in cancer is almost always confined to the squamous cell carcinoma (SQC) domain, independently of the organ of origin22,28–34 and the squamous cell component of adenosquamous carcinoma (ADSQC) in the lung.34,35 Our previous observation that lung AD could overexpress TA p63 but not p40 isoforms at both the IHC and molecular levels (in abstract form only)36 and that p40 was consistently negative in fine-needle aspiration cytology samples of AD37 prompted us to investigate in more detail the diagnostic implications of the duet p40/TTF1 on a consecutive series of biopsy and cellblock samples of NSCLC. We hypothesized that TTF1-negative and/or strongly p63-expressing lung AD22,38 would be a diagnostic dilemma in the event of poor differentiation or inadequate morphology, so additional markers consistently negative in AD (such as p40),36,37 through an innovative algorithm, could prove to be useful tools in challenging cases.

The study was aimed at evaluating the effectiveness of a novel, two-hit IHC model of p40 and TTF1 on limited diagnostic material from 30 AD, 10 SQC, 5 ADSQC, and 1 sarcomatoid carcinoma (SC) to forecast the eventual diagnoses on surgical specimens according to the current lung cancer classification.10,39 In particular, negativity for p40 always excluded by definition SQC and ADSQC and paved the way to the likelihood of facing with AD also in the event of TTF1 negativity. The epithelial-mesenchymal transition, which is particularly developed in SC,40,41 was missed by either marker.

MATERIALS AND METHODS

Patients and Tumors

A series of 46 consecutive preoperative NSCLC samples consisting of 24 fine needle aspiration biopsy cellblocks, 12 fiberoptic bronchoscopy or transcutaneous core biopsies of pulmonary nodules, and 10 transcutaneous core biopsies of regional lymph nodes from 30 males (range, 43–77 years) and 16 females (range, 45–70 years), with the corresponding, paired surgical specimens (31 lobectomies, 14 pneumonectomies, and 1 atypical resection), were raised from the archives of the Department of Pathology and Laboratory Medicine of the IRCCS National Cancer Institute of Milan between 2006 and 2009. The lack of a previous history of cancer elsewhere in the body before sampling was also required for entering the investigation (approved by the institutional review board). Twenty-nine of the 46 patients (63%) underwent histology-guided neoadjuvant chemotherapy (CT) before radical surgery. According to the seventh edition of the TNM staging system,42 there were 4 tumors stage IA (2 CT), 4 IB (3 CT), 5 IIA (3 CT), 8 IIB (4 CT), 21 IIIA (15 CT), 3 IIIB (2 CT), and 1 IV. All biopsies and surgical specimens had been fixed in 4% buffered formaldehyde solution for 12 to 24 hours and embedded in paraffin. No special stains were used to highlight mucous deposition within tumor cells43,44 to test the robustness of our minimalist IHC approach and also because of a lower sensitivity rate of mucin stains in comparison with IHC. All the original hematoxylin and eosin (H&E)-stained sections of surgical specimens were blindly reviewed by two experienced pathologists in lung cancer (G.P. and A.F.) without knowledge of patients’ identity or original tumor categorization according to the 2004 World Health Organization classification criteria on lung cancer,39 assuming that these revised diagnoses made up the gold standard for any comparison. No revision was planned on the original H&E-stained sections of biopsies or cellblocks, inasmuch as the main end point of the study was to verify whether and what extent an innovative diagnostic approach based on the use of two biomarkers only could faithfully parallel the corresponding IHC profiles and, hence, eventual diagnoses on surgical specimens. All tumors were categorized as much definitively as possible, avoiding diagnosing NSCLC-NOS or large cell carcinoma (LCC). In particular, ADSQC was defined by morphology (concurrence of AD and SQC components, either accounting for at least 10%),39 whereas SC was outlined by the presence of sarcomatoid (giant and spindle cell) elements accounting for at least 10%.39 Large cell neuroendocrine carcinoma was excluded by morphology and IHC of appropriate pan-endocrine markers.39 So settled, the study comprised 30 AD, 10 SQC, 5 ADSQC, and 1 SC. The relatively higher proportion of ADSQC we observed in our tumor series was likely to be related to the prevalence of locally advanced stage and poorly differentiated tumors, when considering that such tumors have been reportedly associating with a particular propensity to give rise to local and distant metastases and pursue a more aggressive clinical course compared with conventional lung carcinomas.45–47 No tumor grading was tried on preoperative biopsy/cellblock samples and the 29 patients undergoing preoperative CT, whereas the remaining 17 chemonaive patients, including 9 AD, 6 SQC, and 2 ADSQC, showed all but 2 (1 G1 and 1 G2) poorly differentiated tumors.

Immunohistochemical Evaluation

All tumor samples, either biopsy/cellblock or surgical, were assessed for TTF1 (clones 8G7G3/1 and SPT24), p40 and p63, whereas the 5 ADSQC, 2 TTF1-negative AD, and the case of SC were also characterized for napsin-A, surfactant, cytokeratin 7 (CK7), cytokeratins 5/6 (CK5/6), desmocollin-3 (DSC-3), and vimentin to better unravel glandular (napsin-A, CK7, and surfactant) or squamous (DSC-3 and CK5/6) cell lineage or the epithelial-mesenchymal transition (vimentin), as elsewhere detailed2,8,37,40,48,49 (Table 1). Briefly, 3- to 4-μm-thick sections were reacted for 30 minutes with the relevant antibodies and then incubated with a commercially available detection kit (EnVision FLEX+, Dako, Glostrup, Denmark) following the manufacturer’s instructions and previously refined IHC methods.8 The specificity of all reactions was checked replacing the primary antibody with a nonrelated mouse immunoglobulin at a comparable dilution or using normal serum alone. Positive and negative controls were used as appropriate.

Immunoreactivity was rendered semiquantitatively on a scale from negative to 5+, taken into account the entire tumor area on paraffin-blocked samples and cellular compartmentalization of antibodies (nuclear area for TTF1, p40 and p63; cytoplasm domain for CK5/6, CK7, napsin-A and surfactant; and membrane/cytoplasm labeling for DSC-3). Tumors were considered negative if staining was completely absent in the relevant cells; 1+ cases showed immunoreactivity in up to 10% neoplastic cells, 2+ cases in 11 to 25% neoplastic cells, 3+ cases in 26 to 50% neoplastic cells, 4+ cases in 51 to 75% neoplastic cells, and 5+ cases in 76 to 100% neoplastic cells. This choice was determined by the need of minimizing variability in the slide assessment when trying punctual percentages. For p40, also the intensity of immunolabeling was taken into account, as strong if similar to the internal control represented by bronchial/bronchiolar basal cell layer or low if less intense than seen in the normal internal control.

Statistical Analysis

Qualitative data were compared by Fisher exact test “t” test, Stuart-Maxwell’s test of overall marginal homogeneity (for square tables with >2 rows/columns), and McNemar’s test of overall bias or directional change (comparing the total frequency of cases above the main diagonal of the data table with the total frequency of cases below the main diagonal).50 Unsupervised and supervised hierarchic clustering analysis was performed using Cluster 3.0 software (http://www.eisenlab.org/eisen/) and visualized by using Java TreeView (http://jtreeview.sourceforge.net). The defining features of the diverse clusters corresponded to the different IHC scores used for the relevant markers. Similarity was measured using Spearman rank correlation metric, and clustering was performed by centroid linkage. Contingency analysis and relative mosaic plots were performed by means of JMP IN software (SAS Institute, Inc., Cary, NC), calculating p values through the likelihood ratio test. Mosaic plots indicated graphically the percentage (expressed from 0 to 1) of patients belonging to the different clusters after stratifying for the different tumor subtypes (AD, SQC, ADSQC, and SC). The diagnostic performance of IHC on both biopsy/cellblock samples and surgical specimens was evaluated approaching receiver-operating characteristic (ROC) analysis, comparing curves by Z-test.8,51 For all tests, two-sided p values were taken into account, with a threshold less than0.05 as being statistically significant.

RESULTS

In Table 2, the comparison between original diagnoses on biopsy/cellblock samples and surgical specimens is shown. It emerged that 33 (72%) of original diagnoses had been correctly classified by morphology on biopsy/cellblock samples, whereas the remaining 13 (28%) tumors had been classified as NSCLC-NOS or misdiagnosed as either SQC or AD.

By IHC, unsupervised hierarchic analysis suggested a nonrandom and overlapping distribution of the relevant markers across the tumor series under evaluation (Figure 1A–C). Supervised hierarchic analysis showed, in either type of material, that AD were mostly positive for TTF1 and essentially negative or at the best 1+ score (up to 1–2% positive cells in 5/30 cases) for p40, with p63 exhibiting 1+ to 3+ score, whereas SQC lacked TTF1 and presented with 4+/5+ score for both p40 and p63 (Figure 1B–D). ADSQC in turn showed variable coexistence of AD- and SQC-related profiles, whereas the case of SC was negative for TTF1 and p40 and showed only focal (1+ score) p63 immunoreactivity (Figure 1B–D). Pairwise comparison of each marker between any two samples by unsupervised (Figure 2A) and supervised (Figure 2B) hierarchic clustering resulted in high correlation coefficients, which ranged from 0.92 for p63 to 0.99 for p40 (Figure 2C). The very close and distinct relationship of the clusters with the diverse tumor categories in either type of material was also confirmed by mosaic plot analysis which showed overlapping of the relevant tumor profiling (Figure 3A, B). ROC analysis showed that the values of the area under curve were higher than 0.9 for each marker and relevant diagnosis in both surgical specimens and biopsy/cellblock samples (Figure 4A, B, respectively), with no significant differences after pairing the two types of samples (Supplementary file 1, http://links.lww.com/JTO/A188).

The punctual distribution of the relevant markers in paired biopsy/cellblock samples and surgical specimens according to the semiquantitative IHC scoring system and some representative pictures taken from two cases of AD, either biopsy or surgical specimen are shown in Supplementary file 2 (http://links.lww.com/JTO/A189) and Figure 5A–H, respectively. In AD, p40 was mostly negative (Figure 5H) or detectable in a minority (5/30) of tumors as an erratic (1–2%), 1+ score occurrence of stained tumor cells (Figure 5D) at variance with p63 that was much more represented in the same cases (Figure 5G–C). Moreover, p40 immunostaining intensity was strong in SQC and squamous cell component of ADSQC in comparison with the few AD showing p40 in 1 to 2% tumor cells, where it was low respect to the internal positive control. No differences were seen between any two paired tissue types for all markers but p63 in AD, and TTF1 immunoreactivity was similar in all but two AD by using either clone (Supplementary file 2, http://links.lww.com/JTO/A189). In the normal lung tissue, p40 was confined to the basal cell layer of bronchial/bronchiolar epithelium (Figure 5D–H) and in myoepithelial cells of the bronchial glands, whereas p63 could also be seen in scattered type II pneumocytes especially in areas of nonspecific pulmonary or subpleural fibrosis.

A diagnostic algorithm was then constructed on surgical specimens by crossing p40 and TTF1 according to either positive or negative profile (Figure 6). In particular, 1+ p40-scoring tumors (five cases, all AD) were regarded as being essentially negative in the algorithm, whereas any TTF1 amount was assumed to point to glandular differentiation. Hence, tumors with p40−/TTF1+ profile were all AD, whereas the p40−/TTF1− combination clustered around two AD and the case of SC (featuring a pleomorphic carcinoma composed of AD and spindle and giant cell carcinoma). The p40+/TTF1− profile featured SQC if p40 was by far higher than 50% (75–100%), whereas the five ADSQC expressed p40+/TTF1− profile in two cases (p40 < 50%) and p40+/TTF1+ profile in three cases (any amount for either marker, with mutual exclusion in two cases and occurrence in the same tumor cells in one case). By additional markers (Table 1), the five ADSQC exhibited immunoreactivity for CK7 (all cases), napsin-A (two cases), and surfactant (two cases) in the glandular component and for DSC-3 and CK5/6 in the squamous cell component, whereas the two p40−/TTF1− AD expressed variably CK7 or CK5/6 but not vimentin, and the case of SC was strongly and diffusely positive for vimentin with variable reaction for CK7 and CK5/6.

Overall, 41/46 tumors (89%) were correctly classified by IHC on biopsy/cellblocks in comparison with surgical specimens, including all cases of AD and SQC but only one of the five ADSQC and no case of SC (Table 3). The global figure of IHC-driven diagnoses, however, could be brought to 45/46 (98%) by also adding the four remaining cases of ADSQC, which featured an exclusive SQC (three cases: TTF1−/p40+, with p40 >50%) or AD (one case: TTF1−/p40+, with p40 1–2% tumor cells) component. Only SC actually escaped diagnostic recognition with p40/TTF1 duet. The sensitivity, specificity, and diagnostic accuracy rates of IHC on the biopsy/cellblock samples are presented in Table 4.

DISCUSSION

A main issue of this article, just like an inspiring principle, was to assess the validity of an IHC approach on biopsy/cellblock samples to effectively forecast the corresponding profiling and eventual diagnoses on surgical specimens when applying a novel two-hit, minimalist algorithm based on p40 and TTF1 IHC to simultaneously spare time, economic resources, and diagnostic material. Overall, our findings are in keeping with previously refined diagnostic conclusions drawn by means of a more extended immunohistochemical panel on an independent cohort of NSCLC biopsy specimens.8 The hierarchic clustering approach in light of the current lung cancer classification,39 the area under curve-ROC curve analysis for each marker and relevant diagnoses between the two types of samples, the high correlation coefficients after pairwise comparison for each marker between any two samples, the lack of differences in the immunoreactivity classes for the relevant markers when crossing biopsy/cellblock samples and surgical specimens (Supplementary file 2, http://links.lww.com/JTO/A189), and the overlapping mosaic plots between clusters and the diverse tumor categories in either type of material were all proofs supporting the belief that biopsy/cellblock samples were as reliable as surgical specimens for this two-hit IHC approach to identify different tumor categories, with particular reference for AD, SQC, and a subset of ADSQC. Such a conclusion is worthwhile because it paves the way to novel perspectives in clinical lung cancer trials too, in which the diagnostic assessment carried out by IHC would have the same and perhaps better relevance, reliability, and weight to the patient clinical management as that obtained by morphology alone, especially when considering the general accuracy of IHC to discover diverse cell differentiation lineages in poorly differentiated lung cancer.52 Although it is commonly said that clinical trials have been essentially guided by morphology, the real contribution of IHC is hard to split after this technique has entered the daily diagnostic armamentarium of most pathologists.53 To face the new scenario of dealing with lung cancer diagnoses on limited material,5 the new AD classification by IASLC/ATS/ERS has wisely proposed a stepwise methodology to ultimately classify tumors for clinical purposes, in particular suggesting a precise terminology when using morphology or other diagnostic adjuncts10 (IHC and/or multidisciplinary setting) on small-sized material. As poorly differentiated AD often are associated with shorter survival, more advanced stage54–56 and increased KRAS mutations (an adverse pronosticator for EGFR-based therapy),57 the way to ultimately identify these tumors remains crucial for therapy. Therefore, to validate our diagnostic algorithm, we implemented the minimalist panel approach only in those cases showing challenging or ambiguous phenotypes (ADSQC with p40+/TTF1+ or p40+/TTF1−; AD and SC with p40−/TT1−), paying particular attention not to exhaust the diagnostic material risking eventual molecular study. To this purpose, we would recommend staining gently with hematoxylin all tissue fragments after fixation to recognize them more easily inside tissue bags or inclusion cassettes and place them accurately along the cut surface of the blocks before trimming and immediately cutting a H&E section and five/six additional unstained sections on polarized or any adhesive-treated slides to carry out diagnostic IHC, if any, and subsequent molecular study. Interestingly, we also successfully destined negative IHC-stained sections to FISH or sequencing analysis, if required, to maximize the concept of sparing material and obtaining clinically useful results (data not shown). As p40 was a polyclonal antibody (at variance with all the other antibodies listed in Table 1), we used other commercially available detection kits (Dako Envision Flex and Ventana BenchMark XT biomarker platform system) obtaining the same results in terms of labeling pattern, staining intensity, or number of immunoreactive tumor cells (data not shown).

Although mixing cytoplasmic and nuclear markers (e.g., TTF1/DSC-3 or p63/napsin-A) into antibody cocktails could be an adequate answer in terms of sensitivity and specificity of reaction to reach valuable diagnostic results,2,37 the validation of a minimalist IHC approach to use in challenging cases is warranted especially to preserve tissue for further molecular testing.9,10 The use of nuclear (p40 and TTF1) over cytoplasmic markers for SQC (DSC-3)37 or AD (napsin-A)48,49 resulted particularly advantageous in severely damaged and shrinkaged areas of biopsies, where cytologic details were poor and residual cytoplasmic decoration is difficult to evaluate. As morphology remains a diagnostic cornerstone for a desirable use of only one AD marker and one SQC marker in demanding cases,5,10,11 we propound p40 as a more powerful diagnosticator than p63 (see also Figure 5), which may be widely expressed in up to one-third of lung AD.22,38,58,59 At variance with TTF1, p40 is diagnostically powerful and meaningful when looking at either positive or negative profiles (Figure 6). Conversely, TTF1 is diagnostically meaningful only if positive, whereas, whenever is negative in the setting of poorly differentiated tumors, it may underline diverse tumor subtypes, including SQC, AD, ADSQC, SC, and even salivary gland-type tumors (Supplementary File 3, http://links.lww.com/JTO/A190). We successfully identified all cases of SQC and AD by crossing p40 and TTF1, whereas only 20% ADSQC and none SC were actually diagnosed because of either sampling errors or failure of these marker to highlight epithelial-mesenchymal transition,40,41,60 in turn faithfully mirrored by vimentin IHC.8 In our study, we categorized tumors as much definitively as possible, avoiding diagnosing NSCLC-NOS or LCC. As LCC probably results from poor differentiation and/or tumor sampling incompleteness,2,61 we usually have been sampling the entire tumor mass or, at least, one block per centimeter in the largest size to discover at least focal and more differentiated, conventional features of glandular or squamous cell lineage (data not shown). However, even maintaining the tumor category of LCC, the p40/TTF1 duet is likely to reliably work by highlighting the underlying SQC or AD differentiation on the basis of the diverse immunoreactivity profiles expressed by tumor cells.

We here propound some simple and practical suggestive comments for the interpretation of the diverse p40/TTF1 profiles depicted in Figure 6 (Supplementary File 4, http://links.lww.com/JTO/A191) and also to confirm previously released considerations.2,8,62

TTF1 immunoreactivity points decidedly to AD differentiation (any amount and distribution), provided that p40 is essentially negative (1–2% p40-immunoreactive cells may be observed in a minority of AD but this finding does not contradict such a diagnosis) at variance with p63 that may be deceptively detected, even consistently in up to one-third of AD58,59 (see also Figure 5). Interestingly, ALK-rearranged AD are most often p63 positive63 but they lack p40 (Pelosi et al., manuscript in preparation).

Diffuse and strong p40 expression excludes AD by definition and greatly supports SQC diagnosis if the percentage of immunoreactive cells is by far greater than 50% (usually 75–100%), provided that TTF1 is completely negative (otherwise think of the possibility of facing with ADSQC and search for appropriate morphology). The immunostaining intensity for p40 is strong in SQC and the squamous cell component of ADSQC in comparison with p40-immunoreactive AD, where it is usually low relative to the internal positive control.

ADSQC diagnosis is heralded by p40+/TTF1+ profile or p40+/TTF1− profile (with p40 < 50%). Sampling error rather than biomarker robustness is likely to hamper the final recognition of ADSQC on both surgical specimens64 and biopsies/cellblocks (as in our investigation). Coexistence of glandular and squamous traits within the same tumor cells we observed in our study was previously described in pulmonary65 or esophageal66 carcinomas to testify the possibility of “amphicrine” biphenotypic tumors. The relatively higher proportion of ADSQC39 may be related to the huge prevalence (38/46 cases, 83%) of locally advanced stage or metastatic tumors and of poorly differentiated tumors (44/46 cases, 96%), when considering that ADSQC has been reportedly associating with a particular propensity to give rise to local and distant metastases and pursue an aggressive clinical course.45–47 Furthermore, the ADSQC incidence seems actually to be rising, in close parallel with a greater awareness for composite tumors in the lung67 and the generalized increase of epidemic pulmonary AD.39,64 Further study is needed on a larger caseload of ADSQC to confirm these preliminary data.

Negativity for p40 and TTF1 does exclude by definition SQC of whichever origin and suggests AD or SC (the latter suggested even on biopsy whether vimentin is diffusely and strongly positive in all tumor cells for full-blown epithelial-mesenchymal transition at variance with poorly differentiated NSCLC8), and also sarcoma, melanoma, or mesothelioma has to be ruled out by appropriate, additional markers and clinical working up.

Negativity for p40 and TTF1 plus lack of vimentin excludes confidently SC, melanoma, sarcoma, and to some extent mesothelioma, paving the way to the possibility of facing with pulmonary AD or unsuspected metastases for which a careful clinic integration is mandatory.

In conclusion, this article first provides evidence on the diagnostic performance of a two-hit minimalist IHC approach on biopsy/cellblock samples based on the duet p40/TTF1, for which either positive or negative profiles are meaningful and diagnostically helpful. In particular, we recommend relying routinely on p40 rather than p6358,59 for subclassifying NSCLC to avoid interpreting TTF1−/p63+ AD as belonging to the category of SQC-component containing tumors.

ACKNOWLEDGMENTS

Supported by “Lega Italiana per la Lotta contro i Tumori” (LILT) and is dedicated to the memory of Carlotta, an extraordinarily lively girl, who untimely died of cancer in the prime of life. The authors thank Dr. William D. Travis for useful suggestions.

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